Biosynthesis of UDP-α-N-Acetyl-d-mannosaminuronic Acid and CMP-β-N-Acetyl-d-neuraminic Acid for the Capsular Polysaccharides of Campylobacter jejuni

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in North America and Europe. The exterior surface of the bacterial cell wall is attached to a polymeric coat of sugar molecules known as the capsular polysaccharide (CPS) that helps protect the organism from the host immune response. The CPS is composed of a repeating sequence of common and unusual sugar residues. In the HS:11 serotype of C. jejuni, we identified two enzymes in the gene cluster for CPS formation that are utilized for the biosynthesis of UDP-α-N-acetyl-d-mannosaminuronic acid (UDP-ManNAcA). In the first step, UDP-α-N-acetyl-d-glucosamine (UDP-GlcNAc) is epimerized at C2 to form UDP-α-N-acetyl-d-mannosamine (UDP-ManNAc). This product is then oxidized by a NAD+-dependent C6-dehydrogenase to form UDP-ManNAcA. In the HS:6 serotype (C. jejuni strain 81116), we identified three enzymes that are required for the biosynthesis of CMP-β-N-acetyl-d-neuraminic acid (CMP-Neu5Ac). In the first step, UDP-GlcNAc is epimerized at C2 and subsequently hydrolyzed to form N-acetyl-d-mannosamine (ManNAc) with the release of UDP. This product is then condensed with PEP by N-acetyl-d-neuraminate synthase to form N-acetyl-d-neuraminic acid (Neu5Ac). In the final step, CMP-N-acetyl-d-neuraminic acid synthase utilizes CTP to convert this product into CMP-Neu5Ac. A bioinformatic analysis of these five enzymes from C. jejuni serotypes HS:11 and HS:6 identified other bacterial species that can produce UDP-ManNAcA or CMP-Neu5Ac for CPS formation.


Figure S3. 1 H
Figure S3.1 H-1 H COSY NMR spectra of UDP-ManNAcA (3) produced in H2O with the catalytic activities of non-hydrolyzing C2-epimerase and C6-dehydrogenase from serotype HS:11.Resonances for the hydrogens labeled with a "R" correspond to the ribose moiety of UDP, while those labeled with a "MU" correspond to those of the ManNAcA moiety.Additional details are provided in the text.

Figure S4. 1 H
Figure S4.1 H-1 H COSY NMR spectra of UDP-ManNAcA (3) produced in D2O with the catalytic activities of non-hydrolyzing C2-epimerase and C6-dehydrogenase from serotype HS:11.Resonances for the hydrogens labeled with a "R" correspond to the ribose moiety of UDP, while those labeled with a "MU" correspond to those of the ManNAcA moiety.Additional details are provided in the text.

Figure S5. 1
Figure S5.1 H-1 H COSY NMR spectra of ManNAc (4) produced in H2O with the catalytic activities of hydrolyzing C2-epimerase from serotype HS:6.Resonances for the hydrogens labeled with a "Man" correspond to the N-acetyl-D-mannosamine moiety.Additional details are provided in the text.

Figure S6. 1
Figure S6.1 H-1 H COSY NMR spectra of ManNAc (4) produced in D2O with the catalytic activities of hydrolyzing C2-epimerase from serotype HS:6.Resonances for the hydrogens labeled with a "Man" correspond to the N-acetyl-D-mannosamine moiety.Additional details are provided in the text.

Figure S8. 1 H
Figure S8.1 H NMR and 1 H-1 H COSY NMR spectra of N-acetylneuraminic acid (5) produced from the catalytic activity of the Neu5Ac synthase from serotype HS:6.The reaction was conducted in H2O.(A) 1 H NMR spectra and (B) 1 H-1 H COSY NMR spectra of Nacetylneuraminic acid(5).Resonances for the hydrogens labeled with a ''S'' correspond to the N-acetylneuraminic acid moiety.Additional details are provided in the text.

Figure S9. 1 H
Figure S9.1 H NMR and 1 H-1 H COSY NMR spectra of N-acetylneuraminic acid (5) produced from the catalytic activity of the N-acetylneuraminate synthase from serotype HS:6.The reaction was conducted in D2O.(A) 1 H NMR spectra and (B) 1 H-1 H COSY NMR spectra of Nacetylneuraminic acid(5).Resonances for the hydrogens labeled with a ''S'' correspond to the N-acetylneuraminic acid moiety.Additional details are provided in the text.

Figure S10 .
Figure S10.Mass spectrometry analysis of the reaction catalyzed by the Nacetylneuraminate synthase from serotype HS:6.(A) Reaction conducted in H2O.(B) Reaction conducted in D2O.Additional details are provided in the text.

Figure S11. 1 H
Figure S11.1 H NMR and 1 H-1 H COSY NMR spectra of CMP-Neu5Ac (6) produced from the catalytic activity of the CMP-Neu5Ac synthase from serotype HS:6.The reaction was conducted in H2O.(A) 1 H NMR spectra and (B) 1 H-1 H COSY NMR spectra of CMP-Neu5Ac(6).Resonances for the hydrogens labeled with a "R" correspond to the ribose moiety of CMP, while those labeled with a "CS" correspond to those of the Neu5Ac moiety.Additional details are provided in the text.

Figure S12 .
Figure S12.Mass spectrometric analysis of the reaction catalyzed by the CMP-Neu5Ac synthase from serotype HS:6 in H2O.Additional details are provided in the text.

Figure S13 .
Figure S13.SSN for the non-hydrolyzing C2-epimerase from C. jejuni serotype HS:11.The closest 5000 sequences to the non-hydrolyzing C2-epimerase from C. jejuni serotype HS:11 at a sequence identity cutoff of 59%.The sequences for the functionally characterized nonhydrolyzing C2-epimerases from E. coli K12 and N. meningitidis DSM15465 are shown in pink.The green and blue circles represent the putative non-hydrolyzing C2-epimerases from various Campylobacter species and C. jejuni, respectively.The yellow circle represents the non-hydrolyzing C2-epimerase from serotypes HS:11.

Figure S14 .
Figure S14.SSN for the UDP-ManNAc C6-dehydrogenase from C. jejuni serotype HS:11.The closest 5000 sequences to the C6-dehydrogenase from C. jejuni serotype HS:11 at a sequence identity cutoff of 73%.The sequence for the C6-dehydrogenase from E. coli K12 is shown in pink.The green and blue circles represent the putative C6-dehydrogenases from various Campylobacter species and C. jejuni, respectively.The yellow circle represents C6-dehydrogenase from serotypes HS:11.

Figure S16 .
Figure S16.SSN for the hydrolyzing UDP-GlcNAc C2-epimerase from C. jejuni serotype HS:6.The closest 5000 sequences to the hydrolyzing C2-epimerase from C. jejuni serotype HS:6 at a sequence identity cutoff of 50%.The sequences for the functionally characterized hydrolyzing C2-epimerases are shown in pink.The green and blue circles represent the putative hydrolyzing C2-epimerases from various Campylobacter species and C. jejuni, respectively.The yellow circle represents the hydrolyzing C2-epimerase from serotypes HS:6.

Figure S17 .
Figure S17.SSN for the Neu5Ac synthase from C. jejuni serotype HS:6.The closest 5000 sequences to the Neu5Ac synthase from C. jejuni serotype HS:6 at a sequence identity cutoff of 50%.The sequences for the functionally characterized Neu5Ac synthases are shown in pink.The green and blue circles represent the putative N-acetylneuraminate synthases from various Campylobacter species and C. jejuni, respectively.The yellow circle represents the Neu5Ac synthase from serotypes HS:6.

Figure S18 .
Figure S18.SSN for the CMP-Neu5Ac synthase from C. jejuni serotype HS:6.The closest 5000 sequences to the CMP-Neu5Ac synthases from C. jejuni serotype HS:6 at a sequence identity cutoff of 55%.The sequences for the functionally characterized CMP-Neu5Ac synthases are shown in pink.The green and blue circles represent the putative CMP-Neu5Ac synthases from various Campylobacter species and C. jejuni, respectively.The yellow circle represents the CMP-Neu5Ac synthase from serotypes HS:6.